Discovery and purification of therapeutic proteins that have potential value as pharmaceuticals can be carried out in a research laboratory using materials and methods that are not suitable for large-scale commercial production of pharmaceutical products. To generate pharmaceutical products on a commercial scale, biotechnological manufacturing operations must be robust and scalable without compromising product quality (Gottschalk, 2003, BioProcess Intl 1(4):54-61). Manufacturing processes for pharmaceutical products must provide cost-effective methods, improved product yields, sufficient capacity to meet demand and, ideally, should provide process scalability to respond to fluctuations in demand. Manufacturing processes for therapeutic proteins must develop cost-effective methods for producing large quantities of the protein in a functional form, as well as methods for purifying the protein to generate a pharmaceutical product of suitable purity for its intended use.
“Research-scale” methods of protein purification, also known as “laboratory-scale” or “bench-scale” methods, are often closely linked to the methods that were used to discover and characterize the therapeutic protein. Often, a yield of only micrograms or milligrams of purified protein is sufficient for characterizing and sequencing the protein. Even after an expression system for recombinantly producing a therapeutic protein has been developed, such expression systems are not necessarily suitable to produce the protein on a commercial scale. In addition, research-scale purification methods may use organic solvents, strong acids, or other reagents that are not desirable or practical on a commercial scale and sometimes not permitted in the manufacture of pharmaceutical products. Further, these purification methods may use separation methods such as molecular sieving or high-performance liquid chromatography (HPLC) that are powerful purification methods in the laboratory but are not easily scalable to commercial levels of production.
Pilot scale processes, e.g. fermentation volumes of 10 L to 100 L of a host cell expressing a therapeutic protein, are suitable for further study of the production process or to produce sufficient amounts of a therapeutic protein for early clinical studies, but even pilot scale processes are not always scalable to manufacturing the amounts required for later phase clinical studies.
One approach to increasing capacity in biotechnology manufacturing involves extending the production capacity or efficiency of the microbial expression system. A variety of well-established biological “factories” are available for producing therapeutic proteins. However, since the production of a functional protein is intimately related to the cellular machinery of the organism producing the protein, each expression system has advantages and disadvantages for use in large-scale production of pharmaceutical products, depending on the protein. E. coli has been the “factory” of choice for the expression of many proteins because it is easy to handle, grows rapidly, requires an inexpensive growth medium, and can secrete protein into the medium which facilitates recovery. However, many eukaryotic proteins produced in E. coli are produced in a nonfunctional, unfinished form, lacking glycosylation or other post-translational modifications, as well as formation of proteins with appropriate disulfide bonding and three-dimensional folding. In addition, material produced in E. coli can have endotoxin contamination. Similar constraints are often encountered using Bacillus species as expression systems. Mammalian cell culture systems provide small amounts of eukaryotic proteins with proper glycosylation and folding, but mammalian cell culture systems are expensive, can be difficult to scale up to commercial production levels, can be unstable, and may require the use of animal serum. Insect cell expression systems are fast, relatively easy to develop, and offer good expression levels for mammalian proteins, but can be expensive, only moderately scalable, and can give inappropriate glycosylation. Yeast expression systems are popular because they are easy to grow, are fast and scalable; however, some yeast expression systems have produced inconsistent results, and it is sometimes difficult to achieve high yields.
One yeast expression system that has shown great promise is the methanotrophic Pichia pastoris. Compared to other eukaryotic expression systems, Pichia offers many advantages, because it does not have the endotoxin problem associated with bacteria, nor the viral contamination problem of proteins produced in animal cell culture (Cino, Am Biotech Lab, May 1999). Pichia utilizes methanol as a carbon source in the absence of glucose, using a methanol-induced alcohol oxidase (AOX1) promoter, which normally controls expression of the enzyme which catalyzes the first step in the metabolism of methanol, as a methanol-inducible promoter to drive expression of heterologous proteins. Pichia's prolific growth rate makes it easily scalable to large-scale production, although scale-up challenges include pH control, oxygen limitation, nutrient limitation, temperature fluctuation, and safety considerations for the use of methanol (Gottschalk, 2003, BioProcess Intl 1(4):54-61; Cino Am Biotech Lab, May 1999). Production under current Good Manufacturing Practice (cGMP) conditions is possible with Pichia pastoris, at the scale of 1000L fermentations (Gottschalk, 2003, BioProcess Intl 1(4):54-61).
Another approach to increasing capacity in biotechnology manufacturing is to improve protein recovery and downstream processing of fermentation products. In downstream processing, processes must be adjustable to accommodate changes and improvements in fermentation titer, media composition, and cell viability, while maximizing the productivity of existing capacity (Gottschalk, 2003, BioProcess Intl 1(4):54-61). Recent advances in chromatography and filtration provide significant increases in selectivity, recovery, and offer high capacities and low cycle times to be compatible with large volume and high expression levels of current fed-batch fermentation processes (Gottschalk, 2003, BioProcess Intl 1(4):54-61).
Despite great advances in improving biotechnological manufacturing, no global solutions exist for every protein. The manufacturing process for a specific therapeutic protein requires novel and innovative solutions to problems that may be specific for that protein or family of proteins. Likewise, successful commercial applications often rely on a combination of specific properties of the protein or family of proteins, and the production processes used for manufacturing that protein or family proteins as pharmaceutical products.